Epigenetic mechanisms make gene expression patterns stable by maintaining gene regulatory information through the cell cycle. In some cases, this is thought to occur through heritable modification of chromatin structure. Proteins encoded by the Drosophila Polycomb Group (PcG) genes maintain key patterns of gene expression during development. These genes and their functions are conserved in mammals where PcG. proteins are also linked to many cancers. To understand how these proteins use chromatin structure to maintain gene silencing and to begin to reconstitute the steps in epigenetic inheritance, in vitro biochemical studies of Polycomb group proteins and chromatin will be carried out. First, detailed studies of a key PcG protein, Posterior Sex Combs (PSC), will be carried out to determine how this protein alters chromatin conformation. Mutations which disrupt in vitro activities of PSC will then be tested for in vivo function. Second, PcG-mediated regulation can be very stable. To test the hypothesis that this stability reflects stable interactions between PcG proteins and chromatin, the off rate of PcG complexes from chromatin will be measured under various conditions in vitro. Finally, a major unsolved mystery is how epigenetic regulators such as PcG proteins maintain gene regulatory information through the disruptive events of the cell cycle. To begin to reconstitute passage of epigenetic information through DNA replication, PcG protein-bound chromatin will be replicated in vitro and the fate of PcG protein-chromatin interactions determined. To connect these biochemical results with in vivo phenomena, the association of PcG proteins with chromatin through S-phase and role of DNA replication in the loss of repression upon depletion of PcG proteins in vivo will be determined. Relevance to public health: Epigenetic mechanisms help maintain cell identities through development by making patterns of gene expression stable. They can also lock aberrant regulatory events, such as those that trigger cellular transformation and cancer, into place. Polycomb proteins are thought to be epigenetic regulators of development and are also upregulated in many cancers. This study aims to understand how these proteins function, which will help understand their role in cancer and may eventually make it possible to target them for therapeutic purposes.